The mechanism of DNA chain initiation in animal cells will be studied. The proposed work will examine synthesis of initiator RNA that primes discontinuously synthesized DNA and will be carried out with isolated nuclei of a human lymphoblast cell line. This will include: (1) examination of the lack of discrimination between ribo or deoxynucleotides, (2) consequences of deoxynucleotide misincorporation or ribonucleotide substitution on DNA chain maturation, and (3) the mechanism of chain initiation in the absence of added ribonucleotides. A second proposed area will develop methods for detection and isolation of RNA primer synthetic activity from animal cells. Assays will be based upon the distinctive properties of primer synthesis that dinstinguish it from other activities in the cell. A third area will examine mitochondrial DNA synthesis to determine the origins of multiple ribonucleotides that exist in mitochondrial DNA. This study will examine, in isolated mitochondria, ribonucleotide primed synthesis and nucleotide specificities of endogenous enzymes.